Download SUPPLEMENTARY MATERIAL FOR:

SUPPLEMENTARY MATERIAL FOR:
The Lgr5 Intestinal Stem Cell Signature: Robust Expression of Proposed Markers
of Quiescent '+4' Cells.
Supplementary Table S1
Affymetrix stem cell signature
Supplementary Table S2
Agilent stem cell signature
Supplementary Table S3
The mRNA stem cell signature
Supplementary Table S4
Genes unique to the “Agilent stem cell signature”
Supplementary Table S5
Genes unique to the “Affymetrix stem cell signature”
Supplementary Table S6
Details about MS experiments
Supplementary Table S7
Identified proteins
Supplementary Table S8
Quantified proteins
Supplementary Table S9
The protein stem cell signature
Supplementary Table S10
Genes in the “mRNA stem cell signature” detected but not enriched in MS
Supplementary Table S11
Proteins unique to the “protein stem cell signature”
Supplementary Table S12
The intestinal stem cell signature
Supplementary Table S13
Probe libraries for single molecule mRNA in-situs
Supplementary Table S14
qPCR primer sequences
Supplementary Figure S1
Workflow
Supplementary Figure S2
Evaluation of protein identification
Supplementary Figure S3
Evaluation of protein quantification
Supplementary Figure S4
Technical and biological reproducibility of proteomic analysis
Supplementary Figure S5
mRNA and protein correlation
Supplementary Figure S6
In situ hybridizations of genes showing a gradient along the crypt axis
Supplementary Figure S7
Bmi1 short term lineage tracing
SUPPLEMENTARY TABLE LEGENDS
Table S1. The Affymetrix stem cell signature. Expression for 20,819 genes was detected from which 379 were
found to be significantly enriched in the stem cells (see Materials and Methods)
Table S2. The Agilent stem cell signature. Expression for 13,967 genes was detected from which 291 were found
to be significantly enriched in the stem cells (see Materials and Methods)
Table S3. The mRNA stem cell signature. 384 genes were found to be significantly enriched in stem cells in both
transcriptomics platforms or significant in one and enriched >1.5-fold in the other.
Table S4. Genes unique to the “Agilent stem cell signature”. Details for the 71 genes found to be unique to the
Agilent stem cell signature.
Table S5. Genes unique to the “Affymetrix stem cell signature”. Details for the 54 genes found to be unique to
the Affymetrix stem cell signature.
Table S6. Details about MS experiments. MS information regarding the two biological experiments.
Table S7. Identified proteins. After combining the two biological replicates, 7,967 unique proteins groups were
identified at a FDR of 1.4%. Uniprot accession numbers were submitted to PANTHER Protein classification system
and classified according to their Molecular Function, Biological Process, Cellular Localization and Protein Class
(464 proteins could not be annotated). Further details regarding the protein identification can be found in
Supplementary Figure S1.
Table S8. Quantified proteins. Overall, 6,077 unique proteins groups were quantified. The average of protein
ratios (log2 scale) between both biological is presented. Sequence Coverage, number of quantified peptides per
protein and the Relative Standard Deviations (RSD) are also provided for each separate biological replicate.
Table S9. The protein stem cell signature. Using a cut-off of 1.5-fold consistently in both proteomic replicas, 278
proteins were found to be enriched in the intestinal stem cells.
Table S10. Genes in “mRNA stem cell signature” detected but not enriched in MS. Details for the 27 genes that
were subtracted from the final intestinal stem cell signature due to their equal expression on protein level between
stem cells and daughter cells as measured by MS.
Table S11. Proteins unique to the “protein stem cell signature”. Details for the 59 proteins found to be unique to
the protein stem cell signature.
Table S12. The intestinal stem cell signature. Combining the two transcriptomics data sets and the proteomic data
set resulted in the definition of 510 genes showing a significant enrichment in Lgr5+ve stem cells.
SUPPLEMENTARY FIGURES
Figure S1. Experimental design. A) Lgr5-GFPhigh (stem cells) and Lgr5-GFPlow cells (daughter cells) were
FACS sorted from small intestine of Lgr5-EGFP-ires-CreERT2 mice. B) Two independent platforms, i.e.
Affymetrix and Agilent, were used for gene expression profiling of the two cell populations C) Using the same cell
populations, peptides were differentially labeled, pre-fractionated by using strong cation exchange (SCX) and
analyzed by LC-MS/MS. A second biological replica was performed where labels were swapped. D) By combining
both approaches, a comprehensive list of genes specifically enriched in the stem cells was obtained. Spatial
expression of candidates found by all three methods was examined by RNA in situ hybridization. For one gene
found to be specifically expressed in stem cells, Smoc2, a knock-in mouse for lineage tracing was generated.
Figure S2. Evaluation of protein identification. The number of PSMs (A), number of peptides (B) and sequence
coverage (C) per protein was compared in the two biological experiments and a good correlation was found. In total,
7,967 unique protein groups could be identified (FDR = 1.4 %, Mascot Ion Score>20). More than 75% of the protein
groups were identified on the basis of at least two unique peptides (D) and more than 5% sequence coverage (E).
Detailed information regarding the identification can be found in Supplementary Table S5.
Figure S3. Evaluation of protein quantification. The protein intensity (A) (calculated based on the peptide XICs),
number of quantified peptides per protein (B) and the variability (C) (calculated as the relative standard deviation)
were compared between the two independent biological experiments and a good correlation was found. This resulted
in the robust and precise quantification of 6,077 unique protein groups (4,817 in both replicates). More than 70% of
the proteins were quantified on the basis of at least 2 unique peptides (D) and more than 85% showed errors below
35% (E). Detailed information regarding the quantification can be found in Supplementary Table S5.
Figure S4. Technical and biological reproducibility of proteomic analysis. In order to check the technical
reproducibility, protein ratios derived from two LC-MS/MS runs of the same SCX fraction were compared (A). The
Pearson product-moment correlation coefficient (r=0.821, N=1,450 proteins) indicated a good correlation. Similar
results were obtained when comparing the protein ratios obtained in the two independent biological experiments
(r=0.749, N=4,817 proteins) (B). Protein ratios are represented by two additional variables (Variability and Number
of quantified peptides per protein) to reflect the confidence of the measurements.
Figure S5. mRNA and protein correlation. Protein ratios were matched to their corresponding mRNA levels (log2
scale) using the official gene symbols for Affymetrix (A) and Agilent (B) platforms. The number of genes that could
be correlated and the Pearson product-moment correlation coefficient are indicated for each case.
Figure S6.
mRNA in situs of genes
showing a gradient along
the crypt axis A mRNA in situ
hybridization screen was
performed for the 31
signature genes in the
central overlap (Figure
2B) to explore their spatial
expression pattern. A
subset of genes showed a
gradual expression from
the bottom to the top of the
intestinal crypts. For Cnn3
the gradient could also be
confirmed on protein level
by immunohistochemistry. Figure S7. Bmi1 short term lineage tracing. A-D) LacZ staining of Bmi1-CreER/R26R-LacZ mice, 20hrs after
induction combined with: A) Periodic acid-Schiff staining for goblet cells. B) Magnification of A showing a LacZpositive goblet cell. C) Lysozyme staining for Paneth cells. D) Magnification of C showing a LacZ-positive Paneth
cell. E-H) LacZ staining of Bmi1-CreER/R26R-LacZ mice 2 days after induction. Small trains of cells are visible in
the CBC compartment, the TA compartment and the villus compartment. I-L) LacZ staining of Bmi1-CreER/R26RLacZ mice 3 days after induction